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Original Article

^a Department of Physiology, University of California, Los Angeles School of Medicine, Los Angeles, California 90095
^b Department of Anesthesiology, University of California, Los Angeles School of Medicine, Los Angeles, California 90095
^c Centro de Estudios Cientificos de Santiago and Department of Biology, University of Chile, Santiago, 676470 Chile
^d August Krogh Institute, University of Copenhagen, Copenhagen, Denmark
Dept. of Physiology, UCLA School of Medicine, 10833 Le Conte Avenue, Los Angeles, CA 90095.310-794-9612

fbezanil{at}ucla.edu

When attached outside the voltage-sensing S4 segment of the Shaker potassium channel, the fluorescent probe tetramethylrhodamine (TMRM) undergoes voltage-dependent fluorescence changes (F) due to differential interaction with a pH-titratable external protein-lined vestibule (Cha, A., and F. Bezanilla. 1998. J. Gen. Physiol. 112:391–408.). We attached TMRM at the same sites [corresponding to M356C and A359C in the wild-type (wt) channel] in a deletion mutant of Shaker where all but the five amino acids closest to S4 had been removed from the S3–S4 linker. In the deletion mutant, the maximal F/F seen was diminished 10-fold, and the F at M356C became pH independent, suggesting that the protein-lined vestibule is made up in large part by the S3–S4 linker. The residual F showed that the probe still interacted with two putative quenching groups near the S4 segment. One group was detected by M356C-TMRM (located outside of S3 in the deletion mutant) and reported on deactivation gating charge movement when applying hyperpolarizing voltage steps from a holding potential of 0 mV. During activating voltage steps from a holding potential of –90 mV, the fluorescence lagged considerably behind the movement of gating charge over a range of potentials. Another putative quenching group was seen by probes attached closer to the S4 and caused a F at extreme hyperpolarizations (more negative than –90 mV) only. A signal from the interaction with this group in the wt S3–S4 linker channel (at L361C) correlated with gating charge moving in the hyperpolarized part of the Q-V curve. Probe attached at A359C in the deletion mutant and at L361C in wt channel showed a biphasic F as the probe oscillated between the two groups, revealing that there is a transient state of the voltage sensor in between, where the probe has maximal fluorescence. We conclude that the voltage sensor undergoes two distinct conformational changes as seen from probes attached outside the S4 segment.

Key Words: Shaker K⁺ channel • S3–S4 linker • S4 displacement • fluorescence quenching • gating currents

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This article has been cited by other articles:

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